PROJECT SUMMARY Since the beginning of the AIDS epidemic 35 years ago, much has been learned about how HIV replicates in human immune cells and how persistent HIV infection causes AIDS. However, questions still remain, including the molecular interactions regulating HIV replication in human cells and which interactions can be targeted in order to cure HIV/AIDS. Recent advances in RNA technologies have led to the discovery of an extraordinary number of long non-coding RNAs (lncRNAs) in the human genome. While the functions of these lncRNAs are largely unknown, individual studies show that lncRNAs can act as regulators of gene expression, positively or negatively, through diverse mechanisms. Recently, we used transcriptome deep sequencing (RNAseq) analysis of HIV-1 infected human CD4+ T cells to identify human lncRNAs that are differentially expressed during HIV infection. We then computationally predicted the functions of these lncRNAs. Our preliminary studies now show that HIV-1 replication in human CD4+ T cells is perturbed when we knocked down the expression of some of the identified lncRNAs. Here, we propose using a systems approach to better understand the roles of cellular lncRNAs in HIV infection and to discover candidate lncRNAs as targets for HIV intervention. This project includes two Specific Aims: 1) prioritize cellular lncRNAs involved in HIV-1 infection of human CD4+ T cells; and 2) experimentally validate candidate lncRNAs as targets for inhibiting HIV replication. In Aim 1, we propose to integrate two complementary high-throughput methods to identify the most relevant lncRNAs. In collaboration, we will conduct a large lncRNA siRNA library screen to identify lncRNAs required for HIV-1 infection. We will also expand RNAseq analysis of lncRNAs across additional conditions. In particular, we will treat HIV-1 infected CD4+ T cells with a series of well-known inhibitors targeting different stages of HIV life cycle, to uncover lncRNAs that are involved in inhibiting HIV-1 replication or that are essential for specific stages of HIV replication cycle. Using information gained from these two approaches, we will perform a comprehensive lncRNA function prediction and candidate prioritization utilizing additional orthogonal datasets. In Aim 2, we will first evaluate the impact of highly ranked candidate lncRNAs on HIV-1 replication in human CD4+ T cells, by knocking down lncRNA expression with targeted antisense oligonucleotides. For selected lncRNAs that significantly inhibit HIV-1 replication, we will identify key pathways and biological functions involved in the inhibition. These will be done by coupling computational network modeling with RNAseq analysis which will determine expression changes induced by lncRNA perturbation. We will also evaluate these lncRNAs with additional mechanistic studies including lncRNA subcellular localization and mapping lncRNA interacting proteins and functional subsequences within lncRNA. Together, these analyses will allow us to better understand the functions of lncRNAs and their role in HIV replication, and to identify specific lncRNAs as novel targets for HIV intervention.